The invention relates to a diagnostic assay for determining susceptibility to breast cancer based on the sequence of the 3xe2x80x2 untranslated region of the prohibitin gene.
Breast cancer is the second leading cause of cancer-related deaths of women in North America. A distinction must be drawn, however, between sporadic and familial or inherited breast cancer. Approximately 10% of all breast cancers are currently classified as strongly familial with many of these appearing to be caused by mutations in the hereditary breast cancer genes BRCA1 or BRCA2. However, at least one-third of breast cancers which seem to run in families are not linked to BRCA1 or BRCA2, suggesting the existence of an additional hereditary breast cancer gene or genes. Recently, studies have suggested the possibility that additional genes important for breast cancer development are located on chromosome 17, based on the observation of tumor suppression in breast cancer cells following the introduction of normal human chromosome 17 without the inclusion of active BRCA1 or p53 (Theile, et al., xe2x80x9cSuppression of tumorigenicity of breast cancer cells by transfer of human chromosome 17 does not require transferred BRCA1 and p53 genes,xe2x80x9d Oncogene 10:439-443 (1995)).
The antiproliferative gene prohibitin was discovered using a subtraction hybridization to enrich for mRNAs preferentially expressed in normally proliferating cells compared to regenerating rat liver cells (McClung, et al., xe2x80x9cIsolation of a cDNA that hybrid selects antiproliferative mRNA from rat liver,xe2x80x9d Biochem Biophys Res Comm 164:1316-1322 (1989); Nuell, et al., xe2x80x9cProhibitin, an evolutionarily conserved intracellular protein that blocks DNA synthesis in normal fibroblasts and HeLa cells,xe2x80x9d Mol Cell Biol 11:1372-1381 (1991)). The human prohibitin gene, which maps to chromosome 17 at q21 (White, et al., xe2x80x9cAssignment of the human prohibitin gene (PHB) to chromosome 17 and identification of a DNA polymorphism,xe2x80x9d Genomics 11:228-230 (1991)), was an initial candidate gene for the familial breast and ovarian tumor suppressor locus based on a frequent loss of heterozygosity in this region in familial and sporadic breast cancers (Black, et al., xe2x80x9cA somatic cell hybrid map of the long arm of human chromosome 17, containing the familial breast cancer locus (BRCA1),xe2x80x9d Am J Hum Genet 52:702-710 (1993); Nagai, et al., xe2x80x9cDetailed deletion mapping of chromosome segment 17q12-21 in sporadic breast tumors,xe2x80x9d Genes, Chromosome and Cancer 11:58-62 (1994)). Furthermore, Sato, et al., xe2x80x9cThe human prohibitin gene located on chromosome 17q21 is mutated in sporadic breast cancer,xe2x80x9d Cancer Res 52:1643-1646 (1992), reported four mutations in a highly conserved region of prohibitin exon 4 in an analysis of 23 sporadic human breast cancers. However, positional cloning studies resulted in the identification of BRCA1 rather than prohibitin (Miki, et al., xe2x80x9cA strong candidate for the breast and ovarian cancer susceptibility gene BRCA1,xe2x80x9d Science 266:66-71 (1994)) as a familial breast cancer gene on chromosome 17.
Previous studies of the prohibitin gene from DNA purified from familial breast cancer patients provided no evidence that any of the patients carried a germline change of the protein coding region of prohibitin genomic DNA. It was therefore concluded that there was no relationship between familial/hereditary breast cancer and mutations in the prohibitin protein coding region. Tokino, et al., Internat""l J Oncol 3:769-772 (1993). Additional studies did not identify any somatic mutations in the prohibitin protein coding region in familial/hereditary breast cancers suggesting that the protein coding region is not frequently mutated in breast cancers. Sato et al., Genomics 17:762-764 (1993).
Previous work by Jupe et al. disclosed a diagnostic test for increased susceptibility to sporadic breast cancer. It was reported that individuals who are heterozygous for the two prohibitin alleles (designated as xe2x80x9cnon-Bxe2x80x9d and xe2x80x9cBxe2x80x9d based on sequence variations found in intron 2 and 5) or homozygous for non-B allele would have low risk for developing sporadic cancer. The probability of developing cancer would increase for those who are homozygous for the B-type allele and again for those who have a mutation in the 3xe2x80x2 untranslated region (xe2x80x9c3xe2x80x2UTRxe2x80x9d) of at least one of the B-type alleles. Analyses were reported of breast-cancer derived cell lines and primary breast tumors showing homozygosity for the B-type allele and somatic mutations in the 3xe2x80x2UTR.
Full length prohibitin cDNAs for the BT-20, MCF7 and SK-BR-3 breast cancer cell lines were sequenced, and mutations restricted to the 3xe2x80x2UTR were identified. These three cell lines were arrested in cell cycle progression when full length prohibitin transcript was introduced by microinjection. All of them were also homozygous for the B-allele. Compared to the sequence of the wild type prohibitin 3xe2x80x2UTR, two point mutations were identified for BT-20: G (guanine) to A (adenine) at position 758 and T (thymine) to C (cytosine) at position 814. MCF7 also had two point mutations: G to A at position 236 and C to T at position 729. SK-BR-3 showed 26 base changes including a change of C to T at position 729. Thus, MCF7 and SK-BR-3 both had a change of C to T at position 729. Jupe, et al., xe2x80x9cProhibitin in breast cancer cell lines: Loss of antiproliferative activity is linked to 3xe2x80x2 untranslated region mutations,xe2x80x9d Cell Growth and Differentiation 7:871-878 (1996).
It has now been found, contrary to the teachings of the prior prohibitin work, that this change from C to T at position 729 is the result not of a somatic mutation, but rather the result of a natural allelic variation at this point, i.e., it is a germline polymorphism. Furthermore, it is a germline polymorphism that can be used as a susceptibility marker for breast cancer. Data indicate that the frequency of homozygosity for 729-T appears to be approximately 4-5-fold higher in breast cancer patients than in unaffected females, that 4% of all breast cancers develop in women who are homozygous T/T (which likely make up less than 1% of unaffected women), and that their lifetime risk of developing breast cancer is approximately 50%.
Thus, it has now been found that the prohibitin gene, located on chromosome 17q21 near the BRCA1 locus, exhibits a germline polymorphism in the 3xe2x80x2UTR that can be used as a susceptibility marker for breast cancer.